Control of the synthesis, storage and secretion of bioactive peptides is crucial to normal endocrine and neural function. Over the past several decades, we have delineated the structure of pro-opiomelanocortin (POMC) and detailed the cell-type specific enzymatic steps leading from POMC to its many product peptides. We selected one of these enzymes, peptidylglycine a-amidating monooxygenase (PAM), as a focus of our studies because it is an integral membrane protein with the potential to communicate information about the lumen of the regulated secretory pathway to cytosolic machinery. Our focus on PAM led to the discovery of Kalirin, a GDP/GTP exchange factor (GEF) for small GTP binding proteins of the Rho family, that interacts with the cytosolic domain of PAM; Trio, its closest relative, also interacts with PAM. In addition to their two RhoGEF domains, members of the Kalirin/Trio family have multiple lipid interaction domains, spectrin repeats, SH3 domains, Ig/FnIII domains and a kinase domain. Studies in C. elegans, along with our work on the many splice variants of Kalirin and Trio, ascribe functional significance to the different isoforms. By flanking an exon common to the major splice variants of Kalirin with loxp sites, we created a mouse with a conditional knockout of Kalirin. After excision of this exon in primary pituitary cultures, corticotrope secretion of ACTH in response to secretagogue was doubled. We will test the hypothesis that normal peptide synthesis, storage and secretion require Kalirin. Using lentiviruses or mating with mice in which expression of Cre recombinase is driven by the POMC promoter or the glycoprotein a-subunit promoter, we will eliminate Kalirin expression in corticotropes or in the majority of anterior pituitary endocrine cells. Hormone synthesis, processing and secretion will be evaluated in vivo and in cell culture. The ability of corticotropes lacking Kalirin to respond to restraint stress or injection of lipopolysaccharide will be assessed. The role of Kalirin in the recycling of granule membrane proteins will be assessed. Immature granules represent a choice point for granule biogenesis and a purification protocol will be developed for corticotrope tumor cells. The effects of secretagogue exposure and PAM expression on the composition of immature secretory granules will be evaluated use DIGE. V-ATPase inhibitors rapidly disrupt formation of immature granules at the TGN; by isolating the aberrant vacuoles formed in concanamycin treated AtT-20 cells, components specific to these organelles will be identified. Finally, we will use knowledge of its individual domains to test the hypothesis that Kalirin functions as a modular machine, coordinating multiple aspects of granule biogenesis and release. In particular, the physiological importance of the ability of Kalirin to inhibit iNOS and restore regulated peptide secretion will be assessed in vivo and explored mechanistically in culture. Mutations in the Kalirin gene or changes in Kalirin expression have been correlated with early-onset coronary artery disease, schizophrenia and Alzheimer Disease, making a better understanding of its functions relevant to human health.